The invention relates to a method for preparing an enzymatic hydrolysate containing di- and tripeptides, from a protein mixture, using proteolytic enzymes.
The subject of the present invention is also a di- and tripeptide-rich hydrolysate.
The importance of hydrolysates rich in di- and tripeptides and having a low free amino acid content is widely recognised by numerous scientific studies.
The physiological importance of the intestinal absorption of di- and tripeptides was discovered about thirteen years ago. Thus, the existence of intestinal transport systems for small peptides, different from those for free amino acids, and the greater efficacy of the absorption when the food supply of proteins is achieved from mixtures enriched with small peptides, have been demonstrated by SLEISENGER et al. in an article entitled "Evidence for a single common carrier for uptake of a dipeptide and tripeptide by hamster jejunum in vitro" published in the journal "Gastroenterology", volume 71, pages 76 to 81 (1976).
Compositions rich in di- and tripeptides are of real interest in dietetics not only for the nutrition of infants, convalescents and anaemic individuals, but also for that of patients whose intestinal absorption is altered, such as patients suffering from HARTNUP disease or cystinuria.
Both in individuals suffering from HARTNUP disease and in those suffering from cystinuria, amino acids are normally absorbed by the intestinal mucosa if they are present in dipeptide form (see BRINSON, R. R., HANUMANTHU, S. K., and PITTS, W. M. (1989): "A reappraisal of the peptide based enteral formulas: clinical applications", Nutritional in Clinical Practice 4:211-217; NAYAB, F. and ASATOOR, A. M. (1970): "Studies on intestinal absorption of amino acids and a dipeptide in a case of Hartnup disease", Gut (Journal of the British Society for Gastroentology) volume 11, pages 373-379 (1970)).
Consequently, the invention can for example be applied in hospital diets, in particular in artificial nutrition administered orally, enterally or parenterally, more particularly by intravenous infusion.
Artificial nutrition is essential for patients incapable of normal feeding because of an incapacity due to a physical damage caused by an accident, a surgical operation, an oesophageal trauma or because of a general physiological state which does not permit normal feeding, (coma, burns).
Other possible applications are immunostimulation or animal nutrition, particularly fish feed, or use as growth stimulator.
Various methods are known for preparing protein hydrolysates containing di- and tripeptides: alkaline or acid hydrolysis, enzymatic hydrolysis and chemical synthesis. However, all of these known methods do not enable a hydrolysate containing not less than 75 mole % di- and tripeptides to be prepared from animal and/or vegetable proteins.
Proteins which can be used for manufacturing a hydrolysate on an industrial scale are, by way of example, as follows:
egg proteins (ovalbumin);
milk proteins: casein, whey, lactalbumins, lactoglobulins;
slaughtering blood proteins: blood plasma, serum albumin, decolorised haemoglobin;
products from the fishing and fish-canning industries, and
proteins of plant origin: soybean and lucerne proteins.
Alkaline hydrolysis or acid hydrolysis of mixtures of proteins such as lactalbumin, ovalbumin, whey and casein by means of strong acids or bases is used at high temperature so as to break the chemical bonds. It leads to the production of mixtures which are greatly enriched in free amino acids and often very highly contaminated with salts (Cl.sup.-, Na.sup.+) which are difficult to remove.
The known chemical methods are easy to implement and enable high levels of hydrolysis to be obtained by breaking a large number of peptide bonds. However, they are drastic and cause the formation of undesirable side reactions as well as a decrease in the nutritional value of the proteins through the degradation of essential amino acids.
Thus, alkaline hydrolysis at high temperature (100 .degree. to 110.degree. C.) often generates the formation of lysinoalanine, a very toxic by-product. Moreover, it is difficult to control the degree of hydrolysis of the finished product.
Proteolytic enzymes which may be used for the enzymatic hydrolysis are enzymes of bacterial, fungal, animal and/or plant origin.
The action of one or more of these enzymes on one or more of these proteins leads to hydrolysates containing not more than 50% of di- and tripeptides.
For example, a method for preparing a di- and tripeptide-rich peptide mixture, which may be used in artificial nutrition and in dietetics, by chromatographic extraction of the di- and tripeptides from an enzymatic protein hydrolysate containing 10 to 50% by weight of di- and tripeptides as well as polypeptides which are larger than tripeptides, is known from the document EP-A-0,274,939.
The substrate for the enzymatic hydrolysis consists of one or more animal or vegetable proteins, for example egg, milk, slaughtering blood and/or soybean or lucerne protein. The enzymatic hydrolysate is purified by ultrafiltration. The purified hydrolysate is then loaded onto an ion exchange resin column in order to increase the proportions of di- and tripeptides in the mixture.
In spite of the use of a mixture of enzymes, enzymatic hydrolysate which is not enriched on an exchange resin column contains not more than 50% of di- and tripeptides, not less than 7% of free amino acids as well as an excessively high percentage of ions (see Table 1 on page 8 of the document mentioned).
The hydrolysate is purified on a cation exchange resin column in order to increase the proportions of di- and tripeptides in the mixture and to decrease the salt concentration. This di- and tripeptide enrichment stage has the disadvantage of decreasing the weight yield but also of being troublesome and expensive.
The document EP-A-0,148,072 describes a method for the proteolysis of plasma proteins in order to provide protein hydrolysates suitable for human and animal nutrition by virtue of their acceptable savour and taste. In this method, the blood plasma serving as hydrolysis substrate is obtained by centrifugation of whole blood collected in the presence of an anticoagulant, on slaughtering. A yellow, light phase, the plasma, and a red, heavy phase, the cruor, are obtained by centrifugation in a separator.
Denaturation of the plasma is carried out beforehand by heat treatment of the proteins at a temperature of 70.degree. to 100.degree. C., the pH being maintained at a value of 5 to 7 for less than 30 minutes, so as to increase the proteolysis yield.
Proteolysis of the plasma proteins is then carried out by adding them to an aqueous reaction medium containing thermolysin, in the presence of calcium ions at a pH of 5 to 7 and at a temperature of not more than 80.degree. C.
The proliferation of mesophilic bacteria is avoided by the use of a range of temperatures between 50.degree. and 60.degree. C.
The judicious choice of enzymes and of hydrolysis conditions results in peptides of defined sizes being obtained as required.
The disadvantage of this known method lies, inter alia, in the low weight yield of di- and tripeptides from the proteolysis. Indeed, filtration of the hydrolysate on Biogel P6 reveals the presence of peptides with a molecular mass of less than 2000, that is to say a mixture of free amino acids and peptides containing 2 to 10 amino acids, the di- and tripeptide content remaining very low. In artificial nutrition, however, peptides of more than three amino acids do not provide the kinetic advantages of the absorption described above.